ABSTRACT The survival of Mycobacterium avium subsp. paratuberculosis was studied by culture of fecal material sampled at intervals for up to 117 weeks from soil and grass in pasture plots and boxes. Survival for up to 55 weeks was observed in a dry fully shaded environment, with much shorter survival times in unshaded locations. Moisture and application of lime to soil did not affect survival. UV radiation was an unlikely factor, but infrared wavelengths leading to diurnal temperature flux may be the significant detrimental component that is correlated with lack of shade. The organism survived for up to 24 weeks on grass that germinated through infected fecal material applied to the soil surface in completely shaded boxes and for up to 9 weeks on grass in 70% shade. The observed patterns of recovery in three of four experiments and changes in viable counts were indicative of dormancy, a hitherto unreported property of this taxon. A dps-like genetic element and relA, which are involved in dormancy responses in other mycobacteria, are present in the M. avium subsp. paratuberculosis genome sequence, providing indirect evidence for the existence of physiological mechanisms enabling dormancy. However, survival of M. avium subsp. paratuberculosis in the environment is finite, consistent with its taxonomic description as an obligate parasite of animals.

[Show abstract][Hide abstract]ABSTRACT: Terrestrial sulfidic springs support diverse microbial communities by serving as stable conduits for geochemically diverse and nutrient-rich subsurface waters. Microorganisms that colonize terrestrial springs likely originate from groundwater, but may also be sourced from the surface. As such, the biogeographic distribution of microbial communities inhabiting sulfidic springs should be controlled by a combination of spring geochemistry and surface and subsurface transport mechanisms, and not necessarily geographic proximity to other springs. We examined the bacterial diversity of seven springs to test the hypothesis that occurrence of taxonomically similar microbes, important to the sulfur cycle, at each spring is controlled by geochemistry. Complementary Sanger sequencing and 454 pyrosequencing of 16S rRNA genes retrieved five proteobacterial classes, and Bacteroidetes, Chlorobi, Chloroflexi, and Firmicutes phyla from all springs, which suggested the potential for a core sulfidic spring microbiome. Among the putative sulfide-oxidizing groups (Epsilonproteobacteria and Gammaproteobacteria), up to 83% of the sequences from geochemically similar springs clustered together. Abundant populations of Hydrogenimonas-like or Sulfurovum-like spp. (Epsilonproteobacteria) occurred with abundant Thiothrix and Thiofaba spp. (Gammaproteobacteria), but Arcobacter-like and Sulfurimonas spp. (Epsilonproteobacteria) occurred with less abundant gammaproteobacterial populations. These distribution patterns confirmed that geochemistry rather than biogeography regulates bacterial dominance at each spring. Potential biogeographic controls were related to paleogeologic sedimentation patterns that could control long-term microbial transport mechanisms that link surface and subsurface environments. Knowing the composition of a core sulfidic spring microbial community could provide a way to monitor diversity changes if a system is threatened by anthropogenic processes or climate change.

[Show abstract][Hide abstract]ABSTRACT: Slurry can harbor multiple microbial pathogens among which Mycobacterium avium subsp. paratuberculosis (MAP).•We evaluated the persistence of MAP in soil and infection of soil Acanthamoeba•Infection of amoeba by MAP provides a protected niche for the persistence•As others have suggested, MAP-infected amoeba may act like a “Trojan horse”

[Show abstract][Hide abstract]ABSTRACT: Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.

Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

evaluate the effects of shade, irrigation, and lime application on survival. Exper-iment 2, a pilot study, began in November 1998 with plots at the same locationsand boxes containing soil and pasture at a third site to evaluate the effect ofshade and potential differences between plots and boxes. Experiment 3 wasundertaken at two locations with plots and boxes, was started in November 1999,and was repeated as experiment 4 in January 2000, to examine the seasonaleffects of solar radiation and shade on the survival of the organism.Field sites. Field sites were established in areas of endemicity for ovine para-tuberculosis at Borenore and Carcoar (altitude, 1,000 m above sea level; 33°Slatitude) in the Central Tablelands district and at Camden (70 m above sea level;34°S) in the Sydney district of New South Wales. At Camden there was anexposed unshaded site, as well as a protected partially shaded site on the verandaof a building. An intentionally shaded site (70%) was constructed on the verandawith knitted polypropylene cloth, and a second site on the veranda was totallyshaded (100%). Shaded enclosures (70%), fully covered with knitted polypro-pylene cloth and measuring 10 by 6 by 2.4 m (length by breadth by height,respectively) were constructed in open paddocks on farms at Borenore andCarcoar. Each was surrounded by a secure perimeter fence to exclude livestockand an earth mound to prevent surface water runoff. With a handheld radiometerit was confirmed that 70% of incident solar radiation was absorbed by the wovencloth at each site and that there was little or no UV radiation in the 100% shadetreatment at Camden. The shaded sites at Camden were completely protectedfrom natural rainfall.At Borenore and Carcoar, marker pegs and string lines were used to createpasture plots and square subplots either 1.5 by 1.5 m in triplicate (experiments 1and 2) or 1.1 by 1.1 m in quadruplicate (experiments 3 and 4), within the shadeenclosures and also in unshaded locations on the northern side of each enclosure.Microirrigation sprayers were installed in plots 3, 4, 5, and 6 to provide water for15 min each night to ensure constantly moist soil conditions. To increase soil pH,fine agricultural lime was applied to plots 4 and 5 at rates of 50 and 250 g/m2(0.5and 2.5 tonnes/ha), respectively, immediately prior to application of fecal mate-rial. Very little pasture was present at the start of experiment 1, and fecalmaterial was applied to bare soil, but pasture was allowed to grow during thisexperiment, and this created shade at soil level. By 5 months there was a densecover of grasses, broadleaf weeds, and clover, particularly inside the shadedenclosures. For experiments 2 to 4, pasture was kept ?10 to 15 cm high byregular manual cutting and removal to simulate grazing by sheep. The vegetationwas grass dominant with broadleaf weeds and clover and covered between 40 and85% of the soil surface in shaded plots and 50 to 95% in unshaded plots.Soil boxes composed of expanded polystyrene (58 by 38 by 23 cm) were filledto a depth of 20 cm with soil. A commercial grass seed mixture (couch, 20%;chewing fescue, 10%; perennial ryegrass, 70%) was sown with a light dressing (10g/box) of fertilizer (4.8% nitrogen as ammonium, 5.7% phosphorus, 5.9% po-tassium chloride, 12.6% sulfur, and 12.4% calcium) 7 days before application ofinfected feces so that the grasses would germinate after contamination of theboxes. Boxes were lightly watered to maintain the viability of the grasses, gen-erally at a rate of ?0.5 liter per box per week. The boxes generally had an evencover of grass shoots to 75 mm high by 1 week after contamination with feces. AtCamden in experiments 3 and 4, rainfall in unshaded boxes supported grassgrowth whereas grass was not watered after 3 months and allowed to brown offin the shaded boxes. A drainage tube was fitted to the base of one box inexperiment 2 to enable collection of runoff water.Weather data. Automatic weather data loggers (Easydata Mk4; EnvirondataAustralia Pty. Ltd., Warwick, Queensland, Australia) were installed at the Bore-nore and Carcoar sites (experiments 1 to 3) and also at Camden (experiments 3and 4). These recorded dry bulb air temperature, soil temperature at 1-cm depth,UV radiation (290 to 400 nm), solar radiation (500 to 1,000 nm with correctionto encompass 400 to 3,000 nm), and rainfall. Daily maximum, minimum, andaverage dry bulb air temperature, soil temperature, rainfall, solar radiation, andUV radiation were recorded or derived from these measurements. For experi-ment 2 at Camden, only the daily maximum and minimum dry bulb air temper-atures in the immediate environment of the boxes were recorded.Source and preparation of naturally infected feces. Feces containing M. aviumsubsp. paratuberculosis were collected from groups of sheep on three separateoccasions, namely, just prior to experiments 1, 2, and 3. The feces used inexperiment 4 were from the same sheep as those used in experiment 3 but werestored at ?80°C for about 2 months. The sheep were infected with M. aviumsubsp. paratuberculosis strain BstEII type S1, IS1311 type S (42). Sheep wereTABLE 2. Experimental design, starting levels of contamination, and maximum observed period of survivalExpt no.Starting dateaUnit (no. ofsubplots orreplicates)TreatmentLocation(s)Viable counts of M. aviumsubsp. paratuberculosisat startMaxobservedsurvival(wks)Shade (%)IrrigationLimeVegetationremovedto simulategrazingbPer g offecalmixturePer cm2ofsoil surface1Jan 1998Plot 1 (3)cPlot 2 (3)cPlot 3 (3)cPlot 4 (3)cPlot 5 (3)cPlot 6 (3)Plot 7 (3)Plot 8 (3)Boxes (2)Plot 9 (4)Plot 10 (4)Boxes (2)Boxes (2)Boxes (3)Boxes (3)Boxes (3)Plot 11 (4)Plot 12 (4)Boxes (2)Boxes (2)Boxes (3)Boxes (3)Boxes (3)No7070707070NoNoPartialNo70No70No70100No70No70No70100NoNoYesYesYesYesNoNoAt startNoNoAt startAt startAt startAt startAt startNoNoAt startAt startAt startAt startAt startNoNoNoLowHighNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesOnceYesYesOnceOnceOnceOnceOnceYesYesOnceOnceOnceOnceOnceCarcoar, BorenoreCarcoar, BorenoreCarcoar, BorenoreCarcoar, BorenoreCarcoar, BorenoreCarcoar, BorenoreCarcoar, BorenoreCarcoar, BorenoreCamdenBorenoreBorenoreBorenoreBorenoreCamdenCamdenCamdenBorenoreBorenoreBorenoreBorenoreCamdenCamdenCamden5.00 ? 1067.11 ? 1057.11 ? 1057.11 ? 1057.11 ? 1057.11 ? 1053.34 ? 1043.34 ? 1041.07 ? 1051.63 ? 1053.16 ? 1043.16 ? 1042.10 ? 1042.10 ? 1042.10 ? 1042.10 ? 1042.10 ? 1043.16 ? 1043.16 ? 1042.10 ? 1042.10 ? 1042.10 ? 1042.10 ? 1042.10 ? 104323232263226325.00 ? 1052Nov 19981.20 ? 1065103Nov 19991.58 ? 105212212121228101016241216554Jan 20001.58 ? 105aJan, January; Nov, November.bVegetative cover provided significant shade if not removed.cThese plots received slurry mix. All other treatments received pellet mix.VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2991

Page 4

individually identified; purchased from a farm at Goulburn, New South Wales;housed in a secure animal house; and fed lucerne pellets, lucerne hay, chaff, andoats, and feces were collected as described elsewhere (46). The feces from eachanimal from each day were collected into plastic bags and held at 4°C, or at?80°C if not required for use within a few days. The quantities of feces collectedin the first year were representative of those in later years and are reportedseparately (46). Feces from animals with soft-formed stools were premixed withchaff (4 liters/kg of feces) to obtain discrete masses of feces which were added tothe other feces with some water and additional chaff to obtain a dry, flaky,free-falling pelleted mixture (85% feces by weight), or they were mixed for longerand pellets were broken down by hand to form a slurry mix. The pooled feceswere thoroughly mixed in a large mechanically rotated drum and then dividedinto portions and stored overnight in sealed plastic bags at 4°C prior to contam-ination of sites. Subsamples were retained at ?80°C for later enumeration of M.avium subsp. paratuberculosis cells (see below). The fecal mixtures contained 105to 106viable organisms per g (Table 2). The organisms in both fecal mixturesused in experiments 3 and 4 were enumerated in April 2000, and counts were thesame.Contamination of plots and boxes. Plots were contaminated evenly by handwith pellet mix at a rate of 0.9 to 1.7 kg/m2, being a level of fecal contaminationconsistent with usual sheep stocking rates and equivalent visually to a pellet uponevery few square centimeters of soil surface, or with slurry mix at a rate of 0.7kg/m2. Similarly each box was contaminated evenly with 300 g of fecal pellet mix.The contamination rates applied to soil were in the range of 104to 106viableorganisms per square centimeter (Table 2). The boxes were contaminated in situ,except for experiment 3, where the fecal mixture was applied at Camden and thecontaminated boxes were then transported for 3 h by vehicle to Borenore.Movement during transport caused surface pooling of water and coating of fecalmaterial in some boxes with mud and adversely affected grass seed germination.Sampling from plots and boxes. In each experiment all the subplots of each ofthe field plots were sampled at each time. A galvanized steel wire grid, 1 m2, with1,600 numbered cells each of 2.5 cm2was placed on the ground and alignedcarefully with a fixed marker peg at the corner of each subplot, and randomnumbers were used to select cells for the collection of samples. A fecal pellet wascollected from the cell containing a pellet that was nearest to the selected cell.Vegetation was parted carefully, and after removal of the pellet, a core 1 cm indiameter by 2 cm deep was taken from the soil beneath the pellet by using asterile 10-ml syringe barrel from which the tip had been cut. Soil cores from plotscontaminated with fecal slurry mixture included the slurry mixture on the surfaceof the soil, and separate collection of fecal material was not attempted. Soil coresincluded surface litter, soil, and plant roots to a depth of about 2 cm as well assome aerial parts of plants where these could not be avoided.Boxes in experiment 2 were marked out into two equal segments and used fortwo consecutive collections of pellets and soil beneath them. About 50 ml ofrunoff water was collected weekly from a drainage tube in the base of box 10 afteroverwatering this box. In experiment 3, each box was marked out into three equalsegments (A, B, and C). One segment of each of two (Borenore) or three(Camden) boxes was sampled at each time.At each sampling, two pools of 10 pellets and subjacent soil cores were takenat random from each subplot or box segment for culture. Pellets and soil coreswere pooled in separate containers. At the final sampling of boxes in eachexperiment, between 4 and 16 times the usual number of samples were collectedto increase the probability of isolating low numbers of organisms. Culture resultsfor the pooled samples of pellets and soil were paired to determine whetherviable M. avium subsp. paratuberculosis organisms were present in that subplot orbox segment (culture site) at each sampling time. After primary culture, allsamples were stored at ?80°C to enable enumeration of the organisms in se-lected culture-positive samples. Precontamination samples consisting of twopools of 10 soil cores were collected from representative subplots as negativecontrols for soil inside and outside the shade enclosures, and negative-controlsoil samples were taken from boxes. These samples were all culture negative.Immediate-postcontamination samples were collected from all subplots andboxes to confirm uniform contamination and effective sampling. These sampleswere all culture positive. Sampling of pellets was continued for as long as theywere recognizable as discrete objects. Grass samples were collected with scissors,with careful cutting so as to avoid contamination with feces or soil.Culture methods. Samples were thoroughly mixed prior to subsamples of 2 gbeing taken for culture. Initially mixing was undertaken by hand with a mortarand pestle and scissors to break up plant material, but in most cases a high-speedelectric blender with metal cutting blades was used (41). Cultures were per-formed using a double incubation and centrifugation method to decontaminatesamples and modified BACTEC 12B radiometric medium (Becton Dickinson) aspreviously described (43, 44). Vials were incubated at 37°C for 20 weeks to detectlow numbers of the target organism (45). Identification of M. avium subsp.paratuberculosis was achieved by detection of IS900 by PCR directly from theBACTEC culture medium, with restriction endonuclease analysis of PCR prod-uct to ensure specificity (10). Grass samples were placed in resealable plasticbags, and 250 to 500 ml of saline with 0.1% (vol/vol) Tween 80 was added so thatthe grass was completely covered. The bag was placed on a rocking platform for1 h at room temperature and turned over every 15 min to ensure thoroughwashing of the grass. The washing water was collected and centrifuged at 11,000? g for 20 min. The pellet was then added to a tube containing 10 ml of salineto sediment debris, and the remainder of the procedure was identical to that usedfor culture of feces. Water samples from box 10 in experiment 2 were centrifugedat 11,000 ? g for 20 min, and the pellet was added to saline and cultured asdescribed above.Enumeration of M. avium subsp. paratuberculosis. Unless otherwise stated, fivereplicate cultures, each of 2 g, were undertaken for each sample, and the organ-ism was enumerated by endpoint titration in radiometric culture medium (46).Dilutions were made in phosphate-buffered saline. Rates of contamination of M.avium subsp. paratuberculosis per unit surface area of soil were calculated basedon the results of enumeration of the organism in the fecal mixture and theamount of mixture applied per unit area.Direct PCR analysis of fecal pellets. DNA was extracted from fecal pellets byboiling, purified over a silica column, and examined for IS900 exactly as de-scribed elsewhere (27).Soil analysis. The soil used in boxes was well mixed, and 1-kg samples weresubmitted for analysis. Standard soil samples were collected from plots in Sep-tember 1999, 20 months after liming plots for experiment 1, with the use of acorer 2 cm in diameter by 10 cm in depth. Twelve cores were collected in a gridpattern from each subplot in plots 2, 3, 4, 5, and 6 (36 cores per plot). Sampleswere well mixed before analysis. Surface samples were also collected from theupper 50 mm of selected plots. Soil analyses were performed by Analysis Sys-tems, Incitec Ltd., Port Kembla, New South Wales, Australia, by standard meth-ods: color and texture by observation; pH meter; conductivity meter; colorimetryfor organic carbon, nitrate nitrogen, sulfur (also measured turbidimetrically),phosphorus, and chloride; and atomic absorption spectroscopy for potassium,calcium, magnesium, sodium, aluminum, and iron.In silico analysis of dormancy-associated genes. The Dps protein (DNA bind-ing protein from starved cells) and the relA gene product (GTP pyrophosphoki-nase) are active in survival and dormancy responses of bacteria under starvationconditions, with homologues known in mycobacteria (2, 15). The DNA se-quences for Mycobacterium smegmatis dps (GenBank accession no. AY065628)and Mycobacterium tuberculosis relA (relA gene accession no. Rv2583c, Tuber-culist Web Server, http://genolist.pasteur.fr/TubercuList/) were submitted to theM. avium subsp. paratuberculosis genome database (http://www.ncbi.nlm.nih.gov,accession no. NC 002944). Matching sequences from M. avium subsp. paratuber-culosis were then analyzed in each reading frame for amino acid sequencessimilar to those of Dps and RelA. Alignments were done in GAP with theBLOSUM62 amino acid substitution table (16, 28) through the Bionavigatorfacility, Australian National Genomic Information Service, University of Sydney.Statistical analysis. (i) Assessment of treatment and time effects on the pro-portion of culture-positive samples. For experiment 1, totals of the culture-positive sites for each treatment in weeks 5 to 9 and weeks 14 to 18 wereexpressed as proportions of the corresponding total number of cultured sites. Forexperiments 3 and 4 combined, proportions of culture-positive sites for eachtreatment in weeks 2 to 6 and weeks 8 to 16, and also weeks 20 to 36 for theshaded treatments at Camden, were similarly determined. Mixed-model logisticregression analyses of the proportions were used to assess the fixed effects ofperiods and treatments and their two-factor interactions, with the effects oflocations and the location-period and location-treatment interactions taken asrandom. The fixed effects in experiment 1 were source of contamination (slurrymix or pellet mix), period (5 to 9 and 14 to 18 weeks), shade (nil and 70%), andslurry treatment (control, lime rate, and irrigation), and those in experiments 3and 4 were month of contamination (November or January), period (2 to 6 and8 to 16 weeks, and 20 to 36 weeks for the shaded treatments at Camden), shade(nil and 70% at both sites; 70 and 100% at Camden), and plot type (field, and boxat Borenore). For the latter experiments, the interactions with location involvedonly the periods and treatments common to the locations. The analyses wereperformed using ASReml statistical software (14). All tests were conducted atthe 5% level of significance (P ? 0.05).(ii) Rates of decay of the number of viable organisms. Counts of the numberof viable organisms [log10(counts/gram)] over time were plotted (Prism; Graph-pad Software Incorporated). Linear regressions of log10(counts/gram) on weeksafter contamination were performed for experiments 1 and 2, excluding the datain later weeks, which were statistical outliers. For experiment 4, a linear mixed2992WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.

Page 5

model which comprised a fixed linear term and a random nonlinear term, fittedas a cubic smoothing spline (37), was first fitted. This model showed that therewere two distinct phases of decline, so separate linear regressions were fitted foreach phase. For each regression relation, 95% confidence limits for the predictedmean values were calculated.RESULTSDuration and patterns of survival. Over the first 12 to 18weeks in the experiments, there were generally marked de-clines in the mean proportions of culture-positive sites to lowor zero values (Fig. 1 to 4). No positive results occurred be-tween 18 and 24 weeks in any experiment. However, in exper-iments 1, 3, and 4, culture-positive results occurred at and after24 weeks for some treatments and on earlier occasions follow-ing one or more negative samplings, although the mean pro-portions were usually low (Fig. 1, 3, and 4).In experiment 1, the organism was recovered from plots atboth sites to 32 weeks after contamination. No significant ef-fects of shade, source of contamination, lime treatment, orirrigation on the proportion of culture-positive sites were de-tected (P ? 0.05) (Fig. 1).In experiment 2, which was a pilot study using boxes for thefirst time, the duration of survival of the organism in feces andsoil on unshaded plots was up to 5 weeks, and up to 10 weeksin soil boxes at the partially shaded location. The rate ofisolation from the shaded location appeared to be greater thanthat from the unshaded location (Fig. 2). Grass samples fromthe boxes were culture positive each week up to and includingweek 4. The time for grass samples to reach peak growth index(5 to 8 weeks) was similar to that of fecal pellets, implyingsimilar viable counts of M. avium subsp. paratuberculosis. Run-off water collected from box 10 was culture positive to week 3,and this represented water that had moved through the soilprofile and between the soil and the inside surfaces of the box.As the duration of survival of the organism was considerablylonger in experiment 1, which started in January, than in ex-periment 2, which started in November, and was shorter inunshaded vegetated plots than in partially shaded boxes inexperiment 2, it was hypothesized that differences in theamounts of solar radiation due to season, vegetation, and di-rect shading may have been important. Therefore, experiment3 was started in early November, shade was included as atreatment at two levels (0 and 70%) for plots and boxes atBorenore and three levels (0, 70, and 100%) for boxes atCamden, and this design was repeated as experiment 4, whichstarted about 3 months later, at the end of January (Fig. 3 andFIG. 1. Percentages of culture-positive sites in experiment 1 grouped by shade treatment. Data for the plots at Carcoar and Borenore werepooled. There were no culture-positive sites for week 57, 61, 65, 69, or 72. Solid bars, no shade; striped bars, 70% shade.FIG. 2. Percentages of culture-positive sites in experiment 2 grouped by shade treatment. There were no culture-positive sites for week 29, 33,or 117; there were no samples for weeks 6 and 7 for the no-shade treatment. Results for grass are not shown. Solid bars, no shade, pooled resultsfor the plots at the sites at Borenore and Carcoar; striped bars, 70% shade, results for boxes at Camden.VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2993

4). In the latter experiment M. avium subsp. paratuberculosissurvived for up to 55 weeks in fecal pellets in the shade but formuch shorter periods in unshaded locations.In experiments 3 and 4, there was a significant interactionbetween month of contamination and period: the mean pro-portions of culture-positive sites in November and Januarywere 68.3 and 29.3%, respectively, for weeks 2 to 6 comparedwith 10.2 and 14.2%, respectively, for weeks 8 to 16. OverFIG. 4. Percentages of culture-positive sites in experiment 4 grouped by shade treatment. (A) Plots at Borenore, fecal pellets sampled only toweek 16 in 0% shade and week 10 in 70% shade; (B) boxes at Borenore, fecal pellets sampled only to week 24 in 0% shade and week 12 in 70%shade; (C) boxes at Camden, fecal pellets sampled only to week 32 in 0% shade and week 76 in 70 and 100% shade. Results for grass are not shown.Solid bars, no shade; striped bars, 70% shade; open bars, 100% shade.VOL. 70, 2004 SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2995

Page 8

weeks 2 to 16 the mean proportion of positive sites for 70%shade (56.1%) was significantly higher than that for nil shade(9.3%). At Camden, over weeks 2 to 36 there was a significantincrease of 17.2% in the mean proportion of positive sitesbetween the 70 and 100% shade treatments. At Borenore, overweeks 2 to 16 the mean proportion of positive sites for boxes(38.2%) was significantly higher than that for plots (20.3%).In experiment 3, grass samples from boxes in 70% shade atCamden were culture positive for 4 weeks while those in 100%shade were positive for 10 weeks. The corresponding values forexperiment 4 were 9 and 24 weeks. The organism was notrecovered from grass from unshaded boxes at Camden in ei-ther experiment. There were few positive cultures from grassfrom boxes at Borenore, but survival was found after 9 weeksin 70% shade in experiment 3.A feature of the results for experiments 1, 3, and 4 was thereappearance of culture-positive results after one or more timepoints at which all samples were culture negative (Fig. 1, 3, and4). To provide additional information on this phenomenon,samples from experiment 3 (boxes, 100% shade, Camden)were examined using direct PCR. M. avium subsp. paratuber-culosis DNA was demonstrated in six of six culture-positivesamples from time zero, six of six samples taken at 10 weeks(only three of which had been culture positive), four of fiveculture-negative samples taken at 12 weeks, and six of sixculture-negative samples taken at 32 weeks. Thus, M. aviumsubsp. paratuberculosis cells were present in pellets in mostsamples even though the organism was not cultivable. In eachexperiment the incubation time required for cultures to reachpeak growth index increased over time, consistent with a de-cline in the number of viable organisms. However, in some ofthe cases where the organism was cultured after a previousculture-negative time point, growth occurred more quickly atthe later time point, suggesting an increase in the viable countor recruitment of viable cells from a dormant state.Retrospective enumeration of M. avium subsp. paratubercu-losis in selected culture-positive samples from experiments 1, 2,and 4 was undertaken and confirmed these observations. Therewas an initial phase of rapid decline in viable count lastingseveral weeks to a few months, but thereafter the pattern wasvariable (Fig. 5). In experiment 1 counts were low or 0 from 9to 32 weeks, while in experiment 2 the count was 0 at weeks 7and 8 but rose to 75 at week 9. For experiment 4 there was asignificant spline trend in the mean count over weeks aftercontamination, with a local minimum estimated near week 8and a local maximum near week 18. The estimated increase inmean count between the sampled weeks 6 and 16 was 0.97 ?0.37 logs, which was significant (P ? 0.05) and indicated thatthere were two decline phases (Fig. 5). This increase in viablecount coincided with a reduction in time to peak growth indexfrom 10 to 6 weeks when these samples were cultured origi-nally. There was a small rise in the viable count in experiment2 between weeks 3 and 4 coinciding with a reduction in time topeak growth index from 8 to 6 weeks.Rates of decay of the number of viable organisms in thedecline phases of experiments 1, 2, and 4 (with the week 16data included as part of the second decline phase) were esti-mated by linear regression, and estimates ranged from 0.55 to0.10 logs/week (Fig. 5). When grouped according to the dura-tion of the decline phase, there was an inverse relation (Table3).Weather data. Representative weather data for a 12-monthperiod at Camden are shown in Fig. 6. Rainfall was evenlyFIG. 5. Log10counts of M. avium subsp. paratuberculosis and linearregressions on weeks after contamination. (A) Experiment 1, fecalpellet and soil samples, data from Borenore and Carcoar pooled;(B) experiment 2, fecal pellet samples collected from partially shadedpasture boxes at Camden; (C) experiment 4, fecal pellet samples col-lected from boxes in the 100% shade treatment at Camden. Resultsshown are the counts for the individual samples, the regression linewith 95% confidence limits for the predicted means, and the slope ofthe line ? standard errors.2996WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.

Page 9

distributed at each site, with periodic heavy falls of up to 100mm/week associated with storms. Carcoar and Borenore re-ceived about 700 mm rainfall annually compared to 500 mm atCamden, which was warmer than the other sites. Maximum drybulb air temperatures approached 40°C at Carcoar and Bore-nore and 45°C at Camden, and minima were below 0°C at eachsite. The main factors varying between shade treatments werethe degree of solar radiation and soil temperature. In un-shaded locations total weekly solar radiation levels exceeded200 MJ/m2in summer and were as low as 25 MJ/m2in winter,while total weekly UV levels were 5 to 7 W/m2in summer and0.5 to 1 W/m2in winter. In unshaded plots or boxes soil tem-perature at the 1-cm depth ranged from about 50°C in summerto just above 0°C in winter at Carcoar and Borenore andapproached 60°C in summer at Camden. In 70% shaded plotsand boxes the maximum soil temperature recorded was about40°C whereas in 100% shade it was about 30°C. The diurnalrange of soil temperatures was much less for shaded than forunshaded locations (Fig. 6).Analyses of soils. The soil used in boxes was a dark yellow-brown, light, sandy loam with low organic matter content, a pHof 5.8 to 6.1, and iron levels of 12 to 30 mg/kg (Table 4). Thesoil present in pasture plots at Borenore and Carcoar was abrown clay loam, had a higher organic matter content than thatin the boxes, was slightly acidic (pH 5.7 to 6.7 across plots), andhad iron levels of up to 130 mg/kg. The application of limeresulted in an increase in pH of about 0.4 U for low lime and1.0 U for high lime at Borenore and 0.2 U for low lime and 0.7U for high lime at Carcoar. The high-lime plots at both Bore-nore and Carcoar had a pH of 7.4 in surface samples.In silico analysis of dormancy-associated genes. Regionshighly similar to dps of M. smegmatis and relA of M. tuberculosiswere identified in the M. avium subsp. paratuberculosis genomesequence. The 552-bp DNA sequence (GenBank accession no.AY065628) that codes for the 184-amino-acid Dps proteinfrom M. smegmatis was used to locate the corresponding regionin the M. avium subsp. paratuberculosis genome databasethrough a Blast search. The predicted amino acid sequenceshad 82.5% similarity and 75.6% identity, including a perfectmatch for each of the amino acids thought to be involved in theDNA binding signature of the active protein in M. smegmatis(see Fig. 8) (15).There was a homologue of M. tuberculosis relA in the M.avium subsp. paratuberculosis genome sequence (88% similar-ity over 2,373 bp). The predicted amino acid sequence of M.avium subsp. paratuberculosis RelA excluded amino acids aris-ing from a 6-bp deletion corresponding to bp 49 to 54 in Mtuberculosis but had 96% similarity and 93.4% identity (see Fig.9).DISCUSSIONThe results of this study support those from trials in thenorthern hemisphere with the cattle strain of M. avium subsp.paratuberculosis and confirm that this taxon can be extremelypersistent in nature, with survival for more than 1 year. Unlikeearlier trials where contaminated material was placed in smallcontainers, survival was studied on farms where Johne’s dis-ease is prevalent, in natural pasture plots and in boxes con-taining soil and grass. The presence of soil and pasture pro-TABLE 3. Decay rates of M. avium subsp. paratuberculosis inshaded locations estimated by linear regression of actual countsExptPeriod ofobservation(mo)Decay rate(logs/mo)ShadeSample14240–10–1.50–1.54–92.21.71.00.4Partial100%Partiala100%Pellets and soil from plotsPellets from boxesPellets from boxesPellets from boxesaShaded veranda boxes.TABLE 4. Soil analysisParameterBoxes PlotsExpt 2 Expt 3Borenore CarcoarNo limeaLow lime High limeNo limeaLow limeHigh limepH (water)pH (CaCl2)pH (water), superficial soilOrganic carbon (% C)Sulfate sulfur (KCl40) (mg/kg)Phosphorus, Colwell (mg/kg)Phosphorus, Bray (mg/kg)Potassium (meq/100 g)Calcium (meq/100 g)Magnesium (meq/100 g)Aluminum (meq/100 g)Sodium (meq/100 g)Chloride (mg/kg)Electrical conductivity (dS/m)Nitrogen Kjeldahl (%)Iron DTPAc(mg/kg)6.255.45.84.855.74.96.42.1143090.353.61.40.590.0880.050.171296.15.36.91.91730110.3541.8NTb0.07110.050.17986.65.97.421734120.365.62NT0.08100.060.18756.55.86.7252760.866.81NT0.180.060.2606.766.9242560.857.21NT0.1160.060.24507.26.57.41.742450.747.80.9NT0.09?50.050.19320.5510248.50.212.31.350.060.19820.070.0412.50.5815.53.50.0851.41.20.220.10570.020.02523aMean value for plots 2, 3, and 6.bNT, not tested.cDTPA, diethylenetriamine pentaacetic acid.VOL. 70, 2004 SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS2997

Page 10

vided a more realistic substrate than what could be achieved ina laboratory environment.When M. avium subsp. paratuberculosis in feces becomesmixed with soil, there is a reduction of 90 to 99% in theapparent viable count of the organism. This is probably causedby binding of bacteria to soil particles, which are excluded fromculture by sedimentation during sample preparation (45). At-tachment to soil also occurs with other nontuberculous myco-bacteria (5). The culture method used, in particular the use ofantibiotics and disinfectants during sample preparation, fur-ther reduces the analytical sensitivity of in vitro culture bykilling more than 2 log10M. avium subsp. paratuberculosis cells(32). Thus, estimates of viable count or duration of survival ofM. avium subsp. paratuberculosis based on culture from soil arelikely to be underestimates. The duration of survival assessedin boxes containing soil and grass was comparable to thatFIG. 6. Weather data from Camden for a 12-month period corresponding to experiments 3 and 4. Contamination occurred on 8 November 1999and 31 January 2000. Temperature data are weekly maxima, averages, and minima. (A) Mean weekly dry bulb air temperatures and total weeklyrainfall; (B) weekly total solar radiation; (C) mean weekly soil temperature, no shade; (D) mean weekly soil temperature, 70% shade; (E) meanweekly soil temperature, 100% shade. ?, maximum; I, mean; Œ, minimum.2998 WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.

Page 11

observed in pasture plots, although there were some differ-ences, generally favoring recovery from soil in boxes. This wasprobably explained by the use in boxes of soil with low organicmatter content. It is easier to isolate the organism from suchsoils than from soils of higher organic matter content (45).Boxes were a useful substitute for plots and may be used toadvantage in future studies because they are simple to set upand maintain, soil type can be chosen, and contamination canbe contained.In addition to recoverability from samples and losses duringculture preparation, and assuming log-linear decay, the ob-served duration of survival of microbes also depends on thestarting level of contamination, so we attempted to standardizethis between trials. However, the measurement of decay rateswas also important, because these may be able to be extrapo-lated to situations with different starting levels of contamina-tion.The survival of the organism in fecal material applied to soilwas greatest (55 weeks) in a fully shaded environment and wasleast where fecal material and soil were fully exposed to theweather and where vegetation was also removed. Vegetationprovides shade at the soil surface, and in experiment 1 thisexplained the observation of survival for 32 weeks in plots thatwere not otherwise shaded. In experiment 3 the duration ofsurvival was only 2 weeks in unshaded plots from which vege-tation was removed to simulate grazing by sheep. Moderatedegrees of shade were significantly protective when organismswere most numerous soon after contamination, but over alonger period a higher level of shade was required for signifi-cant protection. Factors such as moisture and soil pH did notappear to influence the duration of survival. Soil pH level hasbeen suggested as a risk factor for Johne’s diseases, throughmechanisms related to iron availability (19). Iron levels in soilsin plots (32 to 129 mg/kg) were higher than those in soils inboxes (12.5 to 23 mg/kg), but survival of M. avium subsp.paratuberculosis was greater in boxes than in plots. This resultmay be due to confounding with soil organic matter content,which was higher in plots than in boxes.Natural rainfall was at times extremely heavy and conceptu-ally may have caused leaching of bacteria from fecal material inall plots and the exposed boxes. However, we were unable tosignificantly reduce the contamination levels in fecal materialin a laboratory trial in which a rainfall event of 400 mm over 4days was simulated by repeatedly soaking pellets in water (datanot shown). Therefore it is unlikely that the organism waseluted completely from fecal material in exposed plots andboxes.M. avium subsp. paratuberculosis was isolated for up to 24weeks from the aerial parts of grasses in this study. Followingseed germination, grass shoots penetrated the surface litterand feces and presumably became contaminated with the or-ganism in this way. The organism may then have been washedFIG. 6—Continued.VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS 2999

Page 12

from grass shoots by rainfall. The shaded boxes at Camdenwere not exposed to natural rainfall and were watered verycarefully by hand, which might explain the higher rate andlonger duration of recovery of the organism from grass atCamden than of that from grass at Borenore.What factors could explain the principal observation fromthis study that survival of M. avium subsp. paratuberculosis wasfavored by shade? Moisture was not a factor promoting sur-vival. Factors apart from moisture that differed dramaticallybetween shaded and unshaded treatments included solar radi-ation, soil temperature, and the diurnal range or flux of soiltemperature. In a recent study of the effect of UV light on thecattle strain of M. avium subsp. paratuberculosis, the organismwas irradiated while suspended in distilled water and appearedto be no more resistant than many other bacterial species (9).The following principles need to be considered: UV radiationcannot penetrate fecal pellets, and therefore it can cause onlysurface disinfection and cannot affect the shaded underside ofpellets; pellets, being dark objects, absorb radiant energy andin turn radiate heat; heat would be conducted to deeper re-gions of the pellet; temperature ranges in pellets on the soilsurface would be greater than those measured in soil at a depthof 1 cm; and evaporation may cool fresh fecal pellets but notdry pellets. Temperature flux stands out as an obvious factorcorrelated with “shade” that could affect survival of M. aviumsubsp. paratuberculosis.Experiments 3 and 4 began with contamination of plots andboxes in early November (presummer) or late January (end ofsummer), respectively. For the first 6 weeks after contamina-tion, the survival rate of M. avium subsp. paratuberculosis inexperiment 3 was more than double the rate in experiment 4.Over the same period, air and soil temperatures in experiment3 were lower and had narrower ranges than those in experi-ment 4 (Fig. 6) but the differences in cumulative solar radiationwere negligible (Fig. 7). These results strongly suggest thattemperature flux influences survival more than solar radiationdoes and support our interpretation that the effect of shade isprimarily through a reduction in temperature flux.The decay rates reported here were estimated from countsfrom fully shaded or partially shaded treatments, as these hada reasonable time series of culture-positive samples. Thesedecay rates are therefore assumed to be the worst-case sce-nario. Although first-order kinetics for log-linear survivalcurves is commonly assumed for microbial inactivation, thereare examples where this is not the case, and tailing of microbialsurvival is sometimes reported (6). Decay rates estimated fromthe linear regressions in this study ranged from 2.2 to 0.4logs/month and were inversely related to the period of obser-vation (Table 3). Differences in environment, climate, andother factors may impact decay rates such that the declinemight occur in a different pattern from that seen in this study.Decay rates for unshaded locations are likely to be higher thanthose for shaded sites. They were inferred from starting countsof M. avium subsp. paratuberculosis in feces, and the observeddurations of survival in feces consequently were highly vari-able, ranging from 1.1 to 7 logs/month. However, when theeffect of dormancy (see below), which led to culture-positiveoutliers, was removed, the decay rates were more consistentFIG. 7. Cumulative solar radiation for experiments 3 and 4 measured at Camden and aligned by week after contamination. I, experiment 3,commencing 8 November 1999; Œ, experiment 4, commencing 31 January 2000.TABLE 5. Decay rates of M. avium subsp. paratuberculosis inpellets in unshaded locations where pasture was either light or wasremoved to simulate grazing, inferred from starting concentrationsof the organism and the observed duration of survival, which wasassumed to be the closest week after the last culture-positivetime pointExptSite SourceStartingconcn inpelletsAssumeddurationof survival(wk)Decay(logs/mo)22333444BorenoreCarcoarBorenoreBorenoreCamdenBorenoreBorenoreCamdenPlotsPlotsPlotsBoxesBoxesBoxesPlotsBoxes1.2 ? 1061.2 ? 1061.6 ? 1051.6 ? 1051.6 ? 1051.6 ? 1051.6 ? 1051.6 ? 105463363551.4 (7)a1.1 (7)1.7 (7)1.4 (5)14 (3)a18 (3)12 (3)14 (1)aFigures in parentheses ignore isolation of low numbers of organisms follow-ing the occurrence of dormancy.3000WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.

Page 13

(range of 3 to 7 logs/month) (Table 5) and greater than thosemeasured for shaded locations. Inclusion of observations of thesmall numbers of viable organisms present following a periodof dormancy is relevant when considering eradication of theorganism from the environment but less relevant when consid-ering control of the infection in livestock. The reliability ofthese inferred estimates for unshaded sites is unclear.In this study M. avium subsp. paratuberculosis was culturedfrom all fecal-soil samples collected soon after contamination,and afterwards there were a progressive reduction in the num-ber of culture-positive samples and an increase in the incuba-tion period required to reach peak growth index. This is con-sistent with a gradual decline in the viability of the organism.However, the time required to reach peak growth index tendedto stabilize, often at around 9 weeks of incubation, with sub-sequent cultures being negative. For soils and feces, incubationperiods to peak growth index greater than about 6 weeks areconsistent with there being only one or several viable organ-isms in the sample (31). Growth index reaching a peak afterthis interval is suggestive of M. avium subsp. paratuberculosiscells requiring a resuscitation phase of several weeks in theculture medium prior to commencement of replication. Afterone or more time points at which all samples were culturenegative, the organism was again recovered from soil and fecalpellets, sometimes with a reduction in the time required forcultures to reach peak growth index compared to that forearlier time points, and in some cases with a sudden increase inthe proportion of culture-positive samples, coinciding with anincrease in viable counts. There are four possible reasons forthese observations: uneven distribution of organisms, system-atic laboratory error, changes in properties of binding of theorganism to feces or soil, and bacterial dormancy.Firstly, consider uneven distribution of fecal material and asampling effect, such that the organism was not included in allsamples. This is unlikely because well-mixed feces were evenlyspread by hand, all postcontamination control samples from allsubplots and boxes were culture positive, the sampling methodwas random and was replicated, and M. avium subsp. paratu-berculosis DNA was demonstrated in numerous samples ofculture-negative fecal pellets. We infer the continuing pres-ence of intact bacterial cells in these pellet samples, as extra-cellular DNA would have been degraded by the ubiquitousDNases from other organisms present in feces.Secondly, systematic laboratory error influencing the sensi-tivity of culture (medium or operator effect) was unlikely be-cause medium controls were used, there was little or no tem-poral overlap in testing batches of samples across the fourexperiments, and both positive- and negative-culture outcomeswere obtained at common test times.Thirdly, a physicochemical effect that causes the organism tochange its binding properties with fecal material or soil com-ponents so that its availability in the culture system changesover time was unlikely within fecal pellets or soil.The fourth explanation is dormancy of M. avium subsp.paratuberculosis cells. The data presented in this study areconsistent with M. avium subsp. paratuberculosis being able toenter a dormant or viable-noncultivable state and later revert-ing to a vegetative form. This phenotypic property has not beenreported before for M. avium subsp. paratuberculosis. Dor-mancy is defined as the state permitting survival of a non-spore-forming bacterial cell without requiring replication. It isgenetically programmed, reversible, and induced by an unfa-vorable environment, classically when an essential nutrient re-quired for growth becomes limiting. Evidence for dormancy isinability to culture the organism until the environment againbecomes favorable and cells regain the ability to divide andthus become detectable (21).In rapidly growing bacterial species dormancy is associatedwith expression of specific genes, at least some of which areknown in mycobacteria. Oxygen depletion of cultures of M.smegmatis (12), Mycobacterium bovis (18), and M. tuberculosis(39) leads to dormancy and increased resistance to antibiotics(40). In M. tuberculoisis prolonged in vitro culture with reducedgrowth rate is associated with expression of heat shock proteinsin the stationary phase of culture (50). Recently, Dps-likeprotein, which confers protection by binding to DNA duringnutritional and oxidative stress in other bacteria, was identifiedin M. smegmatis and a homologue was found in the M. aviumgenome (15). An in silico investigation identified a putativesequence in M. avium subsp. paratuberculosis which containedeach of the amino acid residues that form the DNA bindingsignature in the M smegmatis protein (Fig. 8). A second gene,FIG. 8. Alignment of the amino acid sequences for the Dps-like protein from M. avium subsp. paratuberculosis (M. ptb) and Dps from Msmegmatis (M. smeg) (GenBank accession no. AY065628). Amino acid residues in boldface and underlined are reported to be involved in the DNAbinding signature (15). Symbols: bar, identical; colon, highly related; period, more distantly related; no symbol, unrelated.VOL. 70, 2004SURVIVAL OF M. AVIUM SUBSP. PARATUBERCULOSIS 3001

Page 14

relA, which is active during the stringent response of M. tuber-culosis to amino acid or carbon source depletion (2), is alsopresent in M. avium subsp. paratuberculosis (Fig. 9). Thesefindings add weight to the proposition that M. avium subsp.paratuberculosis is capable of dormancy. However, the stimulusfor dormancy in the present study is unclear apart from sepa-ration of this obligate parasite from its host with consequencesinferred for access to nutrients. Similarly, there must have beenan environment favorable for reversion to the vegetative state,which might have occurred in nature or might have occurredonce dormant cells were added to culture media. However, theculture media alone, which were constant throughout thestudy, were not sufficient to resuscitate dormant cells, as therewere time points in the longitudinal study at which all sampleswere culture negative and later time points at which somesamples were culture positive.FIG. 9. Alignment of the amino acid sequences for the RelA-like element from M. avium subsp. paratuberculosis (M. ptb) and RelA from M.tuberculosis (M. tb) (relA gene accession no. Rv2583c, TubercuList Web Server, http://genolist.pasteur.fr/TubercuList/). Symbols: bar, identical;colon, highly related; period, more distantly related; no symbol, unrelated.3002 WHITTINGTON ET AL.APPL. ENVIRON. MICROBIOL.